PROJECT SUMMARY (See instructions); The aim of this project is to develop new methodology to quantitatively image targeted magnetic nanoparticle (mNP) distribution in vivo in each microscopic compartment to provide mechanistic insights into where targeted mNPs collect and how targeted delivery might be improved. The project will combine two novel synergistic methods, optical ratiometric fluorescence spectroscopy (OFS) and magnetic spectroscopy of mNP Brownian motion (MSB), to quantify bulk concentration uptake and then quantify the level of specific binding to the target ligands in vivo, focusing initially on HER2/neu targeted mNPs which can be internalized when bound. Both measurement techniques proposed here have been uniquely developed at Dartmouth and we have considerable existing resources and expertise that can be leveraged. The global distribution and the bound fraction will be measured with OFS by injection of non-targeted and targeted mNP decorated with different fluorophores. Through quantification of the ratio of uptake, the fraction of bound agent can be measured. There is a large resource of in vivo fluorescence tomography apparatus at Dartmouth, allowing measurement with MicroCT, ultrasound, high field MRI and whole body MRI scanners, and substantial expertise in software, hardware and animal studies have developed in the past decade to allow routine application of this approach. The project will adapt existing hardware/software to couple with magnetic spectroscopy and study the mNP developed by this program. MSB has the ability to measure the binding energies ofthe mNP directly in vivo by evaluating the mNP Brownian motion. Currently no methods are able to do this. The average binding energy for the mNP at each position will be measured. The average binding energy and the bound faction from OFS will provide estimates ofthe fraction of mNP bound to the cell surface and in vesicles. In vivo and ex vivo studies will be used to validate the specificity of the binding, as well as the localization relative to the MSB signal. This technology platform is a fundamentally new, unique way to quantify mNP binding in vivo, and the tools developed here can be utilized in a wide range of mNP targeting assessments. When developed within the program, we can make the technology available for other mNP targeting constructs to help assess the relative success of targeting in vivo, and track the binding status over time. The ultimate value of this technology would be to allow longitudinal tracking of binding in vivo in clinical treatments, thereby providing a custom binding dosimeter for use in patient-specific treatments.